J Mol Biol. 2020 May 1. pii: S0022-2836(20)30323-5. doi: 10.1016/j.jmb.2020.04.026. [Epub ahead of print]
Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context.
Rat V1, Pinson X2, Seigneuret F3, Durand S4, Herrscher C5, Lemoine R6, Burlaud-Gaillard J7, Raynal PY8, Hourioux C9, Roingeard P10, Tramier M11, de Rocquigny H12.
Author information
1
INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé -, BP 3223 37032 Tours Cedex 1, - France. Electronic address: [email protected].
2
Microscopy Rennes Imaging Centre, SFR Biosit, UMS CNRS 3480-, US, INSERM 018, Université de Rennes, 2 Avenue du Professeur Léon Bernard, 35000 Rennes, France. Electronic address: [email protected].
3
INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé -, BP 3223 37032 Tours Cedex 1, - France. Electronic address: [email protected].
4
INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé -, BP 3223 37032 Tours Cedex 1, - France. Electronic address: [email protected].
5
INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé -, BP 3223 37032 Tours Cedex 1, - France. Electronic address: [email protected].
6
Plateforme B Cell Ressources, EA4245 "Transplantation, Immunologie et Inflammation", Université de Tours, 10 Boulevard Tonnellé, 37032 Tours Cedex 1, - France. Electronic address: [email protected].
7
Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé -, BP 3223 37032 Tours Cedex 1, - France. Electronic address: [email protected].
8
Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé -, BP 3223 37032 Tours Cedex 1, - France. Electronic address: [email protected].
9
INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé -, BP 3223 37032 Tours Cedex 1, - France; Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé -, BP 3223 37032 Tours Cedex 1, - France. Electronic address: [email protected].
10
INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé -, BP 3223 37032 Tours Cedex 1, - France; Plate-Forme IBiSA des Microscopies, PPF ASB, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé -, BP 3223 37032 Tours Cedex 1, - France. Electronic address: [email protected].
11
Univ Rennes, IGDR (Institute of Genetics and Development of Rennes) - UMR CNRS 6290, 2 Avenue du Professeur Léon Bernard, 35000, Rennes, F-35000, France; Microscopy Rennes Imaging Centre, SFR Biosit, UMS CNRS 3480-, US, INSERM 018, Université de Rennes, 2 Avenue du Professeur Léon Bernard, 35000 Rennes, France. Electronic address: [email protected].
12
INSERM U1259 MAVIVH, Université de Tours and CHRU de Tours, 10 boulevard Tonnellé -, BP 3223 37032 Tours Cedex 1, - France. Electronic address: [email protected].
Abstract
Hepatitis B virus (HBV) core protein (HBc) is essential to the formation of the HBV capsid. HBc contains two domains: the N-terminal domain (NTD) corresponding to residues 1-140 essential to form the icosahedral shell and the C-terminal domain (CTD) corresponding to a basic and phosphorylated peptide, and required for DNA replication. The role of these two domains for HBV capsid assembly was essentially studied in vitro with HBc purified from mammalian or non-mammalian cell lysates but their respective role in living cells remains to be clarified. We therefore investigated the assembly of the HBV capsid in Huh7 cells, by combining FLIM (Fluorescence Lifetime Imaging Microscopy)/FRET (Förster's Resonance Energy Transfer), FCS (Fluorescence Correlation Spectroscopy) and TEM (Transmission Electron Microscopy) approaches. We found that wild-type HBc forms oligomers early after transfection and at a sub-micromolar concentration. These oligomers are homogeneously diffused throughout the cell. We quantified a stoichiometry ranging from ~170 to ~230 HBc proteins per oligomer, consistent with the visualization of eGFP-containing HBV capsid shaped as native capsid particles by TEM. In contrast, no assembly was observed when HBc-NTD was expressed. This highlights the essential role of the CTD to form capsid in mammalian cells. Deletion of either the third helix or of the 124-135 residues of HBc had a dramatic impact on the assembly of the HBV capsid, inducing the formation of mis-assembled oligomers and monomers, respectively. This study shows that our approach using fluorescent derivatives of HBc is an innovative method to investigate HBV capsid formation.